The overall goal of these studies is to learn more about the detailed mechanism of horse liver alcohol dehydrogenase, in particular about conformational changes that occur when coenzymes bind and when ternary complexes interconvert. The rates of the conformational changes will be determined with various spectral probes using rapid reaction techniques. Since modification of lysine residue 228 increases the activity up to 10-fold, apparently by altering the rate at which enzyme changes conformation, the mechanism of the activation and the rates of the conformational change will be determined. The roles of functional groups at the active site will be elucidated by chemical modification and pH dependency studies. The equilibrium position for the interconversion of ternary complexes ("internal equilibrium"), which is a measure of the "efficiency" of the enzyme will be determined. The pH dependencies, the effects of variation of redox potential of the reactants, and the propensity to bind in "non-productive" modes will be characterized. The size and shape of the substrate binding pocket will be defined in relation to the three-dimensional structure of the protein. Based on knowledge of the structure and mechanism of the enzyme, specific activators will be synthesized and evaluated. One type of activator is a coenzyme analog with functional groups that can methylate the amino group of lysine residue 228. A second type of activator is one that participates in a coupled exchange reaction and oxidizes the enzyme NADH complex, thereby by-passing the rate-limiting dissociation of coenzyme. These activators should be useful for defining the rate-limiting role of alcohol dehydrogenase in ethanol metabolism and eventually for the treatment of acute and chronic alcoholism.